My long-term research goals are to identify and understand the mechanisms of bacterial gene expression in response to changes in the environment. The objective is to determine which gene products of pathogenic organisms allow survival, growth, and reproduction within a hostile host environment. The model system that we study involves an organism that normally inhabits the soil and water, Pseudomonas aeruginosa. Immunocompromised individuals, cancer patients, cystic fibrosis patients, and persons with extensive burns can suffer serious and sometimes fatal infections from P. aeruginosa. In my laboratory we have focused on one of the extracellular virulence determinants produced by P. aeruginosa, exoenzyme S. Exoenzyme S is a member of the ADP-ribosyltransferase family of bacterial toxins with target specificities that include eukaryotic cytoskeletal proteins and small GTP-binding proteins that may mediate vesicle trafficking within cells. The molecular events that include production of exoenzyme S in vivo, intoxication of cells, and the events that damage cells are not understood. However, in animal infection and tissue culture model systems it is clear that exoenzyme S production correlates with epithelial damage and dissemination of Pseudomonas to the bloodstream. The specific aims of this study are to: 1. Analyze the mechanisms regarding exoenzyme S production; 2. Identify the mechanism of exoenzyme S export; and 3. Determine the requirement of exoenzyme S in P. aeruginosa pathogenesis and dissemination. To achieve these aims, my laboratory utilizes both genetic and biochemical approaches to clone, characterize, express, and purify proteins involved in the exoenzyme S pathway. With these strategies we have isolated two structural genes for exoenzyme S, a regulatory locus, and two loci that we propose encode proteins required for exoenzyme S export. This application focuses on the regulatory pathway and the mechanisms that govern exoenzyme S expression, the export pathway as it relates to exoenzyme S regulation, and the role that exoenzyme S plays in P. aeruginosa pathogenesis. The proteins controlling exoenzyme S synthesis and secretion are highly homologous to proteins controlling the expression of virulence in Yersiniae. Thus, the study of the exoenzyme S pathway may be the key to the discovery of novel Pseudomonas extracellular or membrane proteins coordinately regulated with exoenzyme S production that (i) contribute to epithelial damage, (ii) alter host immune response, or (iii) may be candidates for vaccine production.